Compounds for protection of cells

ABSTRACT

This invention is related to a compound with the structural formula (I) 
     
       
         
         
             
             
         
       
         
         
           
             wherein, 
             R1, and R2 are independently selected from the group consisting of C 1 -C 6  alkyl and is preferably methyl, ethyl, propyl or isopropyl; 
             R3 is selected from the group consisting of CH 2 NHR 9 , C(═O)YR 10 , —CH2OH

This invention is related to compounds for the protection of cells, inparticular cultured cells, blood and tissue cells and blood platelets,or thrombocytes. It is further related to compounds for use as or inmedicaments. In addition it is related to a receptacle comprising thecompounds and cells, in particular mammalian cells and mammalian bloodplatelets, for the protection of the cells. Further, the invention isrelated to a method for protection of cells, in particular a method forprotection of cells during storage.

Compounds that protect cells and that are used as a medicament forprotecting cells against, for example oxidative stress-induced celldamage are known in the art. For example tropolone derivatives showneuroprotective activity as described in Koufaki et al. (Eur J Med Chem.2010 March; 45(3):1107-12). Oxidative stress in cells and cell damageare often related with aging and diseases related to aging. Recentstudies revealed that compounds such as nitrones can be used in thetreatment of ischemic stroke and as anti-cancer agents (reviewed inFloyd et al. Free Radic Biol Med. 2011 Sep. 1; 51 (5):931-41).

Other examples of compounds which present cell protective properties, inparticular neuroprotective activity, are hybrids of chroman and catecholmoieties as described in Koufaki et al. (Bioorg Med Chem. 2010 Jun. 1;18(11):3898-909; Bioorg Med Chem. 2009 Sep. 1; 17(17):6432-41), andisoxazole substituted chromans (Bioorg Med Chem. 2011 Aug. 15;19(16):4841-50).

Cell protection is also required during storage of cells. This isespecially the case when cells are cooled down to e.g. 4° C. or −80° C.,and warmed again for use in cell assays or clinical applications.

Blood platelets are cell types that are very difficult to store.Currently, blood platelets are stored under constant agitation at 20-24°C. Storage at room temperature provides an environment where anybacteria, skin flora or other blood or skin borne micro-organisms thatare introduced to the blood component during the collection process mayproliferate, since growth at these temperatures is not limited. Thesecontaminated blood platelets can no longer be used for transfusion. Forthis reason, storage may not be longer than five days, which results inthe fact that more than 15% of the collected blood platelets are expiredbefore they can be used (are/can or were/could). Storage at lowtemperature would prevent bacterial proliferation. However, plateletsshow cold induced platelet storage lesion (PSL), [since thisabbreviation is used later on] when they are cooled, even briefly to 4°C. These platelet cold storage lesions begin to occur even after briefexposure to temperatures less than 20° C. and are even observed inpatients undergoing surgery during which the temperature of the wholebody or of parts of the body is decreased to less than 20° C. Plateletexposure to temperatures of less than 20° C. results in structuralinjury and functional activation of normal platelets. Keycharacteristics of platelet cold storage lesion are (1) reversible toirreversible morphological change from a discoid cell to speculatedspheres with protruding filipodia, depending on the duration of exposureto temperatures less than 20° C. and (2) irreversible immune independentmicroaggregation of platelets by increased cell-cell interaction, (3)membrane clustering of the glycoprotein GPIb on the surface ofplatelets, which is the signal for microphages to remove the plateletsfrom the bloodstream; and (4) subsequent recognition and phagocytosis oftransfused platelets by macrophages upon transfusion into a recipient.Several compounds and their effect on the storage of platelets have beenstudied. For example, U.S. Pat. No. 7,964,339 describes the use ofpolyethylene glycol and derivatives to modify platelets which has aneffect on cold storage. Further, Amorini (Blood Transfus. 2007 January;5(1):24-32) showed that a glucose solution may have a positive effect onthe storage of blood platelets. U.S. Pat. No. 6,833,236 and EP patentno. 1 221 835 B1 describe the use of trehalose for protection ofthrombocytes when they are freeze-dried or dried in an FTS dryer, forstorage of the blood platelets.

Despite this knowledge of compounds which have an effect on the storageof blood platelets, there is a further need to elaborate new compoundswhich have a positive effect on the storage of blood platelets. Inaddition, it is further required to find new compounds which protectcells against cell damage in vitro and in vivo, where the cell damagecan be caused by oxidative stress, among others.

It is an object of this invention to provide compounds that can be usedto protect cells, such as mammalian cells, against cell injury.

It is another object to provide compounds that can be used to protectblood platelets against cold storage lesion.

It is further an object to provide compounds that provide protection ofcells against damage caused by several medical indications, such asindications involved by aging diseases, indications involved byoxidative stress, and/or indications involved by the formation of ablood clot.

These objects and other objects are solved partially, if not, completelyby a compound as described in the attached claim 1.

In particular, these objects and other objects are solved partially, ifnot completely, by a compound with the structural formula (I)

-   -   wherein,

R1, and R2 are independently selected from the group consisting of C₁-C₆alkyl and is preferably methyl, ethyl, propyl or isopropyl;

R3 is selected from the group consisting of CH₂NHR₉, C(═O)YR₁₀, —CH2OH,

where * indicates the point of attachment of R₃ to the remainder of themolecule;

R₄, R₅, R₆, R₇, R₈ are independently selected from the group consistingof H, —OH, alkyl, substituted alkyl, preferably hydroxyalkyl, aryl,substituted aryl, halogen, oxygen, heteroaryl, substituted heteroaryl;preferably wherein R₇ is not aryl,

X is selected from the group consisting of H, ═O, ═S;

Y is selected from the group consisting of O, NH, S;

R₉ is selected from the group consisting of hydrogen, alkyl, substitutedalkyl, preferably hydroxyalkyl, alkenyl, aryl, heteroaryl;

R₁₀ is selected from the group consisting of alkyl, substituted alkyl,preferably hydroxyalkyl or cyanoalkyl, aryl, OH;

R₁₁ and R₁₂ together with the atom N to which they are attached form asaturated or unsaturated 3, 4, 5, 6, 7 or 8 membered ring, incorporatingone or more additional, such as one, two, or three N, O, or S atoms;

R₁₃ and R₁₄ together with the atom N to which they are attached form asaturated or unsaturated 3, 4, 5, 6, 7 or 8 membered ring, optionallysubstituted with alkylalcohol, and in case that R₃ is CH₂NHR₉,C(═O)YR₁₀, —CHOH or

than is R₁ and R₂ isopropyl.

In one embodiment of the present compounds, R₇ or R₁₀ is not OH if Y isO.

In one embodiment, the present compound is not5-[4-[N-[(2RS)-6-hydroxy-2,5,7,8-tetrametylchroman-2-ylmethyl]-(2S)-pyrrolidine-2-methoxy]phenylmethylene]thiazolidine-2,4-dione.

In one embodiment, the invention is related to the present compounds foruse as a medicament.

In one aspect, the invention is further related to the use of a compoundin treatment or prophylaxis of ischemic stroke, cerebral seizure,thrombosis, embolism, hemorrhage, cardiovascular disease, arthritis,diabetes, cancer, in particular cancer related to aging,atherosclerosis, heart failure, myocardial infarctions, schizophrenia,bipolar disorder, fragile X syndrome, sickle cell disease, and chronicfatigue syndrome, chronic obstructive pulmonary disease (COPD), aneurodegenerative disease such as Alzheimer disease, Parkinson disease,Lou Gehrig's disease, Huntington's disease, hypothermia/reperfusioninjury, hemorrhagic shock, aging, hypertension, renal failure due tovarious kidney diseases, asthma, inflammatory bowel disease, hepatitisand liver cirrhosis, migraine, hyper-homocysteinemia, infection diseasesinvolved in attacking thrombocytes, such as haemorhorragic fever, inparticular ebola and chagas wherein the compound has the structuralformula of (I)

-   -   wherein,

R1, and R2 are independently selected from the group consisting of C₁-C₆alkyl and is preferably methyl, ethyl, propyl or isopropyl;

R3 is selected from the group consisting of —CH2OH, CH₂NHR₉, C(═O) YR₁₀,

where * indicates the point of attachment of R₃ to the remainder of themolecule;

R₄, R₅, R₅, R₇, R₈ are independently selected from the group consistingof H, —OH, alkyl, substituted alkyl, preferably hydroxyalkyl, aryl,substituted aryl, halogen, oxygen, heteroaryl, substituted heteroaryl;

X is selected from the group consisting of H, ═O, ═S;

Y is selected from the group consisting of O, NH, S;

R₉ is selected from the group consisting of hydrogen, alkyl, substitutedalkyl, preferably hydroxyalkyl or substituted hydroxyalkyl,alkylbenzylfluoride, alkenyl, aryl, substituted aryl, preferablyhaloaryl, heteroaryl;

R₁₀ is selected from the group consisting of hydrogen, alkyl,substituted alkyl, preferably hydroxyalkyl or cyanoalkyl, haloalkyl,alkylamide, substituted alkylamide, aryl, substituted aryl, preferablynitrobenzyl, halobenzyl, alkylbenzoyl, OH, alkenyl, alkadienyl,alkylhalide, arylhalide, —CH₂(C═O)O-alkyl, heteroaryl, substitutedheteroaryl, —NH—CH₂CH₂CN;

R₁₁ or R₁₂, alkyl, substituted alkyl, preferably an alkylamine, or formtogether with the atom N to which they are attached a saturated orunsaturated 3, 4, 5, 6, 7 or 8 membered ring, optionally incorporatingone or more additional, such as one, two, or three N, O, or S atoms,optionally substituted with an alkyl, alkylalcohol;

R₁₃ and R₁₄ together with the atom N to which they are attached form asaturated or unsaturated 3, 4, 5, 6, 7 or 8 membered ring, optionallyincorporating one or more additional, such as one, two, or three N, O,or S atoms, optionally substituted, preferably substituted with analkyl, alkylalcohol;

The compounds of general formula (I) containing an assymetrical centreare of isomeric form. The racemic and enantiomeric forms of thesecompounds also form part of this invention.

The inventors surprisingly found that the compounds having the formulaas described above protect cells against cell damage. The inventorsdeveloped several new compounds as described above, and found that theyare able to protect cells against cell damage. Cell damage can haveseveral causes and is established under stress conditions. Cell damagemay eventually lead to cell necrosis or apoptosis. The inventorsperformed tests on several cell types and surprisingly found that theabove compounds have an effect on the cells and protect the cell againstcell damage or cell injury under stress conditions. With stressconditions is understood oxygen deprivation (hypoxia and ischemia);occurrence of physical agents (such as mechanical trauma, extremes oftemperature, burns and deep cold, sudden changes in atmosphericpressure, radiations, electric shock); occurrence of chemical agents anddrugs; occurrence of infectious agents, immunologic reactions; geneticdiseases; nutritional imbalances, such as injury, infection, cancer,infarction, poisons, occurrence of ROS (Reactive Oxygen Species), andinflammation. Compounds according to the invention can be used as amedicament to protect cells against the above mentioned causes of cellinjury.

In one embodiment, the invention is related to the compounds asdescribed above for use in treatment of oxidative stress, inflammation,derailment of proteostasis, DNA damage (e.g. irradiation), calciumoverload, poisons/toxic agents, derailment or errors of metabolisminduced cellular damage.

In another embodiment, the invention is related to the compounds asdescribed above for use in treatment or prophylaxis of diseases that arerelated to aging, neurodegenerative diseases, infection, diabetes andother indication in which cell damage is involved.

In another embodiment, the invention is related to the compounds asdescribed above for the treatment or prophylaxis of conditions relatedto aging or oxidative stress, in particular ischemic stroke, cerebralseizure, thrombosis, embolism, hemorrhage, cardiovascular disease,arthritis, diabetes, cancer, in particular cancer related to aging,atherosclerosis, heart failure, myocardial infarctions, schizophrenia,bipolar disorder, fragile X syndrome, sickle cell disease, chronicfatigue syndrome, chronic obstructive pulmonary disease (COPD) andneurodegenerative diseases such as Alzheimer disease, Parkinson disease,Lou Gehrig's disease and Huntington's disease, tissue damage mediated bya viral or bacterial infection.

In another embodiment, the invention is related to a compound asdescribed above for use in treatment of ischemia/reperfusion injury.

In another embodiment, the invention is related to a compound asdescribe above for use in treatment of indications involved withoxidative stress induced cell damage.

In one aspect, the inventors also found that the compounds as describedabove protect blood platelets and prevent blood platelets to adhere oraggregate, and prevent to undergo the shape-change.

In another embodiment, the invention is related to a compound for use inthe treatment or prophylaxis of disorders leading to or caused by aplatelet disfunction such as arterial thrombosis, arterial fibrillation,pulmonary embolism (PE), deep vein thrombosis (DVT), or venousthromboembolism (VTE), congestive heart failure, stroke, myocardialinfarction, genetic or acquired hypercoagulability, or platelet defectscaused by haemorhorrgic fevers such as ebola, Marburg disease andchagas.

In a preferred embodiment, the compound for use in the treatment orprophylaxis of disorders leading to or caused by a platelet disfunctionis chosen from Sul 100, Sul 117, Sul 118, Sul 120, Sul 121, Sul 125, Sul126, Sul 132, Sul 136, Sul 138, Sul 139, Sul 141, Sul 142, Sul 143, Sul144, Sul 145 (see table 1 for IUPAC names).

In another aspect, the invention is related to a solution comprising thecompounds as described above and cells. The inventors found that thecompounds protect the cells against cell injury, which may eventuallylead to cell death via necrosis or apoptosis. The compounds according tothe invention provide protection against cell injury. The protection canbe provided during storage. Cells stored with compounds according to theinvention have a decreased cell death compared with cells stored withoutthe compound.

In one embodiment, the above compounds protect mammalian cells, such ascultured cell lines (e.g. from human origin), stem cells, primary cells,blood platelets, blood cells and tissue cells. The cell lines can bebrought in culture for the manufacture of viral vaccines, biologicalproducts produced by recombinant DNA technology in the cell cultures,such as proteins, hormones, enzymes, antibodies, etc.

In another embodiment, the invention is related to a medium comprisingone of the above compounds, in which cells which are grown to form atwo- or three dimensional cell culture. In addition, the compounds canbe used to protect cells that are used for tissue engineering.

Preferred compounds according to this embodiment are compounds selectedfrom the group consisting of those listed in Table 1.

TABLE 1 Code Chemical name SUL-083 2,2,5,7,8-pentamethylchroman-6-olSUL-084 (S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acidSUL-085 (R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acidSUL-089 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide SUL-090N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-091N-butyl-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-0926-hydroxy-N-isopropyl-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-093(E)-N-(3,7-dimethylocta-2,6-dien-1-yl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-095(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(morpholino)methanone;SUL-097N-(4-fluorobenzyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;SUL-0986-hydroxy-N-((S)-2-hydroxy-1-phenylethyl)-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-1006-hydroxy-2,5,7,8-tetramethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide; SUL-1016-hydroxy-N,2,5,7,8-pentamethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide; SUL-1026-hydroxy-2,5,7,8-tetramethyl-N-(3-(piperidin-1-yl)propyl)chroman-2-carboxamide; SUL-1046-hydroxy-2,5,7,8-tetramethyl-N-(3-nitrophenyl)chroman-2-carboxamide;SUL-106N-(4-fluorophenyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;SUL-107 methyl4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamido)benzoate; SUL-108(4-butylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone; SUL-109(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone; SUL-110((2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone; SUL-111N-((R)-2-amino-2-oxo-1-phenylethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-112(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone; SUL-114N-(2-bromoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;SUL-115N′-(2-cyanoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carbohydrazide;SUL-1162-(((4-fluorobenzyl)amino)methyl)-2,5,7,8-tetramethylchroman-6-ol;SUL-117 2-((butylamino)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1186-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid; SUL-1192-(hydroxymethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol; SUL-1206-hydroxy-N-((R)-1-hydroxypropan-2-yl)-2,5,7,8-tetramethylchroman-2-carboxamide SUL-121(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanoneSUL-122 (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)methanone; SUL-123N-(2-cyanoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide;SUL-124 6-hydroxy-N-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-125(R)-N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-126(S)-N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; SUL-1282-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1292-((((S)-2-hydroxy-1-phenylethyl)amino)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1302,5,7,8-tetramethyl-2-(piperidin-1-ylmethyl)chroman-6-ol; SUL-131N,6-dihydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxamide; SUL-132(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone; SUL-133(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone; SUL-1342-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1352-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol; SUL-1362-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid; SUL-137(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(piperazin-1-yl)methanone; SUL 138(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone; SUL-1392-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)acetic acid; SUL-140 ethyl2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetate; SUL-141(S)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid; SUL-142(R)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid; SUL-143(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid; SUL-144(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid; SUL-145(2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid;

A preferred embodiment according to the invention is a medium comprisingblood platelets and one of the above compounds.

In a preferred embodiment, blood platelets are protected against and areprevented to adhere or aggregate, or to undergo shape change by theaddition of Sul 100, 117, 118, 120, 121, 125, 126, 132, 136, 138, 139,141, 142, 143, 144, 145.

In another aspect, the invention is related to a method for protectionof cells comprising adding a compound as described above to a cell.Preferably, the present method is an ex vivo method. Accordingly, thecompound can be used for protection during storage of cells. The storagecan occur at a temperature which is suitable for storage of theparticular cell and can be at and below 37° C., preferably between −80and 37° C., such as between 10-25° C.; 0-10° C., about 4° C.; between−20 and 0° C. and between −80 and −20° C., at room temperature, −80° C.etc. The inventors found that the above compounds protect the cellsagainst cell injury during and after cooling down, and especiallyagainst injury that occurs during warming up back to the functionaltemperature. More cells survive these stress conditions and thus thestorability increases when the cells are stored together with thecompound according to the invention, compared with cells that are storedwithout the addition of a compound according to the invention.

In one embodiment the medium, which can be a typical platelet storagebuffer or additive solution, comprises a compound according to theinvention having a concentration that is between 1. 10⁻³-1.10⁻³⁰M,preferably about 1.10⁻⁶, 1.10⁻⁷, 1.10⁻⁸, 1.10⁻⁹, 1.10⁻¹⁰M.

In another aspect, the invention is related to a method for storage ofcells, wherein the cells are blood platelets comprising adding acompound according to the invention to the blood platelets. As describedabove, blood platelets are stored at room temperature, before they canbe used for transfusion. Storage of blood platelets at 4° C. results inrapid loss of platelet viability and function. The inventors found thatwhen blood platelets are stored at a temperature below 20° C., e.g. at4° C. with a compound according to the invention, the storability ismuch higher and can therefore longer be stored compared with bloodplatelets stored without the compounds. The compound according to theinvention can thus be used to obtain an increased storability of bloodplatelets during cold storage. Moreover, when a compound is added to theblood platelets, the aggregation and adhesion of the blood plateletsdecrease. Moreover, platelets stored with a compound according to theinvention may still show an aggregation response upon stimulation withADP or epinephrine. The compounds according to the invention help tomaintain the functionality of the platelets.

The invention is further related to a method for protection of bloodplatelets against platelet storage lesions. This means that a compoundaccording to the invention is involved in the process which affectsmorphological changes of platelets when they are cold stored. Thecompound according to the invention provides a reduced shape change, adecrease in coagulation and has an effect on the release of granulecontents, exocytosis of cytosolic proteins or on the glycoproteinpatterns on the platelets. The compounds according to the invention havean influence on the platelet activation mechanism, and provide adecrease in PSL (platelet PSL=platelet storage lesion), or decreasedamount of activated platelets, subsequently leading to a decrease ofapoptosis. The above compounds preserve the platelet function of bloodplatelets after having been stored in a cold environment.

In this aspect, preferred compounds are Sul 100, Sul 117, Sul 118, Sul120, Sul 121, Sul 125, Sul 126, Sul 132, Sul 136, Sul 138, Sul 139, Sul141, Sul 142, Sul 143, Sul 144, Sul 145.

The compounds according to the invention better preserve the capacity ofplatelets to aggregate or to adhere upon stimulation, even when theyhave been stored at 4° C.

In one embodiment, the invention provides platelets stored with acompound according to the invention for use as platelet transfusion.

In one embodiment the blood platelets are derived from platelet-richplasma (PRP). In another embodiment, the platelets are derived from abuffy coat (BC) or the apheresis method. Platelets for transfusion canbe prepared by three different methods: (a) the platelet-rich plasma(PRP) method; (b) the buffy coat (BC) method; and (c) the apheresismethod. Studies comparing PRP and BC platelets have shown no differencein the in vitro quality of such platelet concentrates when they arestored for up to 5 days at room temperature. In apheresis of plateletsor plateletpheresis the platelets are derived from one specific donor.The three methods are well described and are known by the person skilledin the art.

In one embodiment, the present invention relates to the use of thepresent compounds, preferably Sul 109, for increasing the cold ischemictolerance of transplant organs, preferably hearts. In other words, thepresent invention relates also to the use of the present compounds,preferably Sul 109, for storing transplant organs, such as hearts.

The technical effects and advantages of the various embodiments andaspects of the methods of the invention correspond mutatis mutandis tothose described for the products of the invention and vice versa.

This then generally describes the invention but to assist withunderstanding, reference will now be made to the accompanying comparisonand non-limiting examples and figures which show embodiments of theinvention.

FIGURE DESCRIPTION

FIG. 1: Schematic overview of hypothermia/reperfusion injury inducingtest where the compound of the invention is first added to the cells at37° C., incubated for 1 h, cooled at 4° C. during 24 h, rewarmed andtested at 37° C.

FIG. 2: Trypan Blue absorption assay. NOD is used as a positive control.Sul 112 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone), Sul 121((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl) (piperazin-1-yl)methanone)Sul 127 (methyl 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate), Sul136(2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid), Sul 89 (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), Sul85 (((R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Sul141((S)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid), Sul 142((R)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid). Sul 136 is a racemic mixture. Sul 141 and Sul 142 arerespectively the S and R isomer. The compounds were added to SMAC cellsat different concentrations. The absorption assay was performed afterrewarming until 37° C. 4° C. means that the cells were first cooleduntil 4° C. C37° C. and C4° C. are tests of SMAC cells wherein the cellsare maintained at 37° C. and first cooled at 4° C. and than rewarmeduntil 37° C., respectively without the presence of compounds.

EXAMPLE 1: PRESERVATION OF HEK CELLS Material and Method:

Human Embryo Kidney (HEK) 293 cells were cultured in DMEM cell culturemedium (Life Technologies, 41965-052) supplemented with fetal calfserum, penicillin and streptomycin. Cells were seeded at a density of0.8-1.2E6 mL-1 in 25 cm2 polystyrene flasks, placed in a 37° C.C02-regulated humidified incubator and were allowed to proliferate for24 hours before commencing experiments.

The compounds SUL-090(N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-091(N-butyl-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-092(6-hydroxy-N-isopropyl-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-093((E)-N-(3,7-dimethylocta-2,6-dien-1-yl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-095 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(morpholino)methanone), SUL-097(N-(4-fluorobenzyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-098(6-hydroxy-N-((S)-2-hydroxy-1-phenylethyl)-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-100(6-hydroxy-2,5,7,8-tetramethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide),SUL-101(6-hydroxy-N,2,5,7,8-pentamethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide),SUL-102 (6-hydroxy-2,5,7,8-tetramethyl-N-(3-(piperidin-1-yl)propyl)chroman-2-carboxanide), SUL-104(6-hydroxy-2,5,7,8-tetramethyl-N-(3-nitrophenyl)chroman-2-carboxamide),SUL-106(N-(4-fluorophenyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-107 (methyl4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamido)benzoate), SUL-108((4-butylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone), SUL-109((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone), SUL-111(N-((R)-2-amino-2-oxo-1-phenylethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-oarboxamide),SUL-112 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone), SUL-114(N-(2-bromoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-117 (2-((butylamino)methyl)-2,5,7,8-tetramethylchroman-6-ol),SUL-118 (6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylicacid), SUL-120(6-hydroxy-N-((R)-1-hydroxypropan-2-yl)-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-121 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone), SUL-122((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)menthanone), SUL-123(N-(2-cyanoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-124(6-hydroxy-N-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-125 ((R)-N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide),SUL-126 ((S)-N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide,SUL-128(2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol),SUL-129(2-((((S)-2-hydroxy-1-phenylethyl)amino)methyl)-2,5,7,8-tetramethylchroman-6-ol),SUL-130 (2,5,7,8-tetramethyl-2-(piperidin-1-ylmethyl)chroman-6-ol),SUL-131(N,6-dihydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxamide),SUL-132 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone), SUL-134(2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol),SUL-135(2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol),SUL-136(2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid), SUL-137 ((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(piperazin-1-yl)methanone), SUL 138((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone),SUL-139(2-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)aceticacid), SUL-140 (ethyl2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetate),SUL-141((S)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid), SUL-142((R)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid), SUL-143((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid), SUL-144((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid), SUL-145((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid were tested.

The compounds were dissolved in dimethyl sulfoxide (DMSO) to severalconcentrations. Before commencing experiments, this stock solution wasdissolved in pre-warmed DMEM and further diluted to obtain aconcentration range from 10 nM-1 mM. The hypothermia rewarming injuryprotocol was used as follows and is summarized in FIG. 1. Cell culturemedium was then replaced by these dilutions and cells were incubated inthe presence of the compound for 1 hour. After incubation, lids wereclosed tightly and flasks were placed in a 4° C. room and cooled for 24hours. After this cooling period, cells were placed back in the 37° C.incubator with lids closed and were allowed to rewarm for another 24hours.

Assessment of Viability

After this rewarming period, microscopic images were taken to assesscell morphology (Nikon D5100, Nikon Diaphot-TMD). Cell culture mediumwas collected and centrifuged (4 minutes, 2000 rpm). The supernatant wascollected and pH was immediately measured to prevent equilibration ofthe pH by the carbonate buffer in the medium, while the pelleted cellswere resuspended in 5 mL of PBS. Cells remaining in the flask werewashed twice with phosphate buffered saline (PBS) while the washingproduct was discarded. Subsequently, the cells were trypsinized byaddition of 0.5 mL of trypsine and pooled with the resuspended pellet.This cell suspension was then stained at a final concentration of 0.2%trypan blue (Sigma, T8154), followed by manual assessment of viabilityand cell number on a Bürker-Türk hemocytometer. To conclude the assay,cell culture medium glucose levels were measured (Roche Accutrend Plus)as an indication of metabolic activity. All of these steps wereperformed with care to prevent mechanical stress to influence or alterthe viability of the cells after cooling.

Results

Table 2 shows the results of the amount of cells that survived theprocess of cooling and rewarming with the addition of the compoundaccording to the invention in different concentration. The amountrepresents the % cells that survived. The number of the compoundcorresponds with the compound as described in table 1. In the control,the same procedure was applied to two badges of HEK cells. The mediumdid not comprise a compound according to the invention. The amount ofcells that survived depends on the used concentration and on the type ofcompound.

TABLE 2 10 100 1 10 100 1 nM nM μM μM μM mM Mean SUL - 17 91.95 93.1091.77 95.58 95.34 94 SUL - 83 0.00 79.38 70.12 55.74 1.11 41 SUL - 846.22 19.79 70.08 95.74 94.50 57 SUL - 85 1.23 0.00 1.43 84.88 78.26 33SUL - 89 34.90 88.63 91.61 87.57 76 SUL - 90 8.67 91.34 90.25 77.9220.41 58 SUL - 91 2.28 82.44 89.53 87.54 0.00 52 SUL - 92 3.13 64.5887.14 86.47 1.55 49 SUL - 93 97.05 96.08 84.04 21.69 8.00 61 SUL - 9498.63 97.31 96.35 89.02 0.00 76 SUL - 95 2.76 86.75 90.38 92.09 85.86 72SUL - 96 5.62 96.34 93.53 96.88 0.00 58 SUL - 97 0.00 95.11 96.37 91.625.17 58 SUL - 98 90.99 97.81 97.54 88.49 1.02 75 SUL - 99 54.44 87.9893.75 69.18 100.00 81 SUL - 100 77.78 76.87 63.25 68.77 55.61 0.00 57SUL - 102 73.24 95.77 93.72 81.97 73.41 93.33 85 SUL - 103 99.74 98.1096.85 98.31 10.27 0.00 67 SUL - 104 99.26 99.49 97.52 98.91 40.91 52.1781 SUL - 105 94.72 95.01 80.50 96.11 32.86 50.64 75 SUL - 106 77.7876.87 63.25 68.77 55.61 0.00 57 SUL - 107 4.42 95.74 92.72 94.77 37.6228.92 59 SUL - 108 0.00 40.89 94.15 96.81 96.57 0.00 55 SUL - 109 92.6498.05 97.01 94.93 93.73 87.37 94 SUL - 111 90.86 81.62 97.33 93.69 94.3479.21 90 SUL - 112 96.26 96.52 95.38 92.97 93.06 0.00 79 SUL - 114 81.1487.16 89.94 87.70 77.93 0.00 71 SUL - 117 0.72 89.29 93.56 84.79 90.090.00 60 SUL - 118 13.67 45.70 36.22 52.88 88.50 92.45 55 SUL - 120 17.6565.27 95.54 91.63 88.73 81.46 73 SUL - 121 87.31 88.97 94.75 88.94 92.740.00 75 SUL - 122 5.15 22.54 85.60 85.17 88.50 55.56 57 SUL - 123 68.4290.50 83.16 88.41 88.16 0.75 70 SUL - 125 8.09 0.00 97.27 96.65 88.4422.50 52 SUL - 126 0.00 0.00 68.12 92.68 98.62 80.21 57 SUL - 127 0.000.00 97.56 96.67 89.19 92.05 63 SUL - 128 28.21 100.00 100.00 100.0099.41 0.00 71 SUL - 129 0.00 5.65 88.80 89.92 91.52 0.00 46 SUL - 1300.00 12.73 85.65 89.61 86.55 1.49 46 SUL - 131 2.99 1.59 22.52 91.1075.38 0.00 32 SUL - 132 93.96 91.41 88.30 91.11 89.73 80.51 89 SUL - 13487.43 86.34 87.74 86.58 88.82 36.51 79 SUL - 135 71.94 83.90 93.82 93.8583.46 33.06 77 SUL - 136 99.60 100.00 98.37 99.52 99.35 100.00 99 SUL -137 78.17 86.12 93.88 90.88 78.71 0.53 71 SUL - 138 92.84 91.74 77.0191.71 93.30 86.94 89 SUL - 139 13.33 4.57 4.26 90.05 89.67 80.33 47SUL - 140 8.70 17.95 95.65 97.00 94.63 0.00 52 SUL - 141 58.51 96.250.75 99.44 96.77 99.12 75 SUL - 142 0.00 73.02 98.51 99.16 99.42 91.8977 SUL - 143 30.59 35.00 14.94 49.47 97.83 98.43 54 SUL - 144 4.08 18.2721.57 25.93 91.01 92.59 42 SUL - 145 12.99 28.68 21.43 7.84 78.61 88.0840 SUL - 146 0.96 17.00 90.63 80.65 85.49 0.00 46 DMSO 0.00 0.54 0.620.00 0.00 0.00 0 Control 4.71 0.00 2

EXAMPLE 2: PRESERVATION OF SMAC CELLS

Rat smooth muscle aortic cells (SMAC cells) were cultured in DMEM cellculture medium (Life Technologies, 41965-052) supplemented with fetalcalf serum, penicillin and streptomycin. Cells were placed in a 37° C.C02-regulated humidified incubator and were allowed to proliferate for24 hours before commencing experiments. The compounds Sul 84, Sul 85,Sul 89, Sul 112, Sul 121, Sul 127, and Sul 136 were dissolved in DMSO toa final concentration of 100 mM. This solution was then dissolved inDMEM cell culture media and added to the cells at differentconcentrations, preincubated during 1 hour at 37° C. cooled down to 4°C. and kept at 4° C. during 24 h. The cells were rewarmed during 1 huntil 37° C. and were tested. A Trypan blue test was performed, asdescribed above for the HEK cells. In addition to this Trypan blueexclusion assay, an absorption assay was performed. The extend of Trypanblue absorption was used as an indication of the number of non-viablecells. Trypan blue was added to a 6-wells plate to a final concentrationof 0.05% and cells were incubated at 37° C. for 5 minutes. Subsequently,excess dye was removed by carefully washing wells three times with coldPBS. After washing, 150 μL of 1% SDS was added to the wells to lyse thecells and free the trypan blue from any non-viable cells. Cell lysateswere centrifuged and supernatant was subsequently transferred to a96-wells plate. 1% SDS was used as a blank and absorption was measuredat 595 nm. Cell death was expressed as a percentage of untreated 4° C.controls (100%).

FIG. 2 shows the results of a Trypan blue absorption assay to evaluatethe amount of cells that survived the hypothermia and rewarming process.The viability of the SMAC cells was up to 90%. Sul 136 is a racemicmixture. Sul 141 and Sul 142 are the R and S isomer, respectively. The Senantiomer has a better effect than the R enantiomer or the racemicmixture, even at lower concentration.

EXAMPLE 3: PRESERVATION OF BLOOD PLATELETS Collection Via PRP

PRP platelets were harvested and 2.1 ml was suspended in Poly Propylenetubes of 5 ml, closed with a stop.

Several compounds according to the invention were added to the bloodplatelets directly after preparing the PRP plasma and incubated during10 minutes at 37° C. or 30 minutes at room temperature. The bloodplatelets with the compound were then stored at 4° C.

A control was stored under shaking conditions at room temperature.Another control was stored at 4° C. without the addition of a compound.The compounds that were added are those listed in table 3.

A sample was taken out of each tube after 264 hours after addition ofthe compound and tested as follows:

-   -   The color and coagulation properties were visually analysed.    -   The thrombocyte cells that survived were counted and it was        evaluated whether the thrombocytes were aggregated.    -   It was evaluated if the thrombocytes still were able to function        after stimulation by the addition of ADP or under influence of        collagen in a 96 well plate.    -   The adhesion was evaluated, meaning the ability of the platelets        to bind collagen in a 6 well plate preincubated with collagen.    -   Via an ELISA assay, it was evaluated whether thromboxane was        secreted from the platelets. Thromboxane facilitates aggregation        and is produced by activated blood platelets.

Table 3 provides an overview of the results of the addition of thecompounds to the blood platelets and evaluates the aggregation of thethrombocytes after addition of the compound according to the inventionand cooling until 4° C.

TABLE 3 Name of compound (see also Final Table 1 for Concentration ofAggregation of blood the iupac Purity of compounds added to plateletsafter name) compound bloodplatelets storage SUL-100 >95%   30 mM +SUL-117 92% 30 mM + SUL-118 100%  30 mM + SUL-120 90% 30 mM + SUL-12190% 30 mM + SUL-125 90% 30 mM + SUL-126 90% 30 mM + SUL-132 99.6%   30mM + SUL-136 94% 30 mM + SUL-138 98.5%   30 mM + SUL-139 90-99%   30mM + SUL-141 97% 30 mM + SUL-142 91% 30 mM ++ SUL-143 98% 30 mM +SUL-144 >90%   30 mM + SUL-145 95% 30 mM + − means that most of thethrombocytes were aggregated a + indicates that a few cells were stillviable but some cells aggregated a ++ indicates that almost all cellswere viable

Collection Via the Apheresis Method and PRP

Platelets were obtained via standard plateletpheresis procedures eitheron Haemonetics (donor 2611811 and donor 2611855) or on Cobe instruments(donor 2611770). According the protocol all units would be obtained withplateletpheresis procedure with the Cobe instrument, but from the threedonations two were not passing the blood bank quality control criteria.Nevertheless, all platelet donations have a similar concentration and asimilar platelet quality. In none of the platelet concentrates clotswere found.

The freshly harvested platelets are weighed on a balance and forsampling of platelet concentrate a small PVC bag was seal-docked to themain platelet concentrate bag. Standard homogenization of plateletconcentrate has to be done with a roller before sampling.

300 μM suspension of Sul 136 is added with a procedure, which keeps thebag content sterile and the Sul 136 is kept in suspension by frequentwhirling and stored at 37° C. before it is added to the freshlyharvested platelets. The Sul 136 suspension is added to three plateletbags directly from the bag. After addition, the content of the plateletbag is gently mixed. After 2 hours the first sample is drawn with aseal-docked sample bag. The sample bag is disconnected by welding andfrom this sample bag the samples are taken for hematology analyzing,aggregometer tests and the flow-cytometry tests. Swirling in the bag isreported by experience of the laboratory worker in the standard format.Platelets without Sul 136 put on a flatbed shaker and stored at roomtemperature were used as control. Another control is stored at 4° C.Platelet samples in which Sul 136 is added, are stored without shakingin a refrigerator at 4° C.

Samples are taken after 2, 24, 48, 96, 168, 216, 264 hours and arestored for 7 weeks.

The samples are measured as follows:

Annexin V Test:

On a Beckman Coulter FC 500 flow cytometer using the Roche Annexin-VFLUOS Staining Kit

Procedure:

A mix of 20 μl Annexin-V fluorescein from Roche kit into 1 ml incubationbuffer was made and 20 μl propidium iodide solution was added. 2 ml ofplatelet concentrate was centrifuged on standard Eppendorf centrifugeand the supernatant was discarded.

1 ml PBS buffer was added and cell concentration was checked whether itwas around 1.000.000. This was diluted 100 times with the preparedAnnexin-V fluorescein and propidium iodide mix and incubated for 15minutes. 500 μl incubation buffer from kit was added and cell analysiswas made in a flowcytometer.

Platelet Activation with ADP and TRAP

Platelet activation was performed on a Beckman Coulter FC 500flowcytometer using CD 41 PE, CD 62p FITC, IgG1 FITC/IgG1 PE antibodiesof a Beckman Coulter kit.

Test Tube 1:

40 μl Dilution buffer was brought in about 15 minutes to 37° C.; 10 μlIgG FITC/IgG PE+10 μl CD41 PE was added and mixed with Vortex, incubated5 min at room temperature; 1000 μl cold HBSS buffer was added;measurement in flowcytometer was performed.

Test Tube 2:

40 μl Dilution buffer was brought in about 15 minutes to 37° C.; 10 μlCD41 PE+5 μl CD62 FITC was added and mixed with Vortex, incubated during5 min. at room temperature; 1000 μl cold HBSS buffer was added;measurement in flowcytometer was performed.

Test Tube 3:

36 μl Dilution buffer+4 μl ADP was brought in about 15 minutes to 37°C.; 10 μl CD41 PE+5 μl CD62 FITC was added and mixed with Vortex,incubated during 5 min. at room temperature; 1000 μl cold HBSS bufferwas added; measurement in flowcytometer was performed.

Test Tube 4:

36 μl Dilution buffer+4 μl TRAP was brought in about 15 minutes to 37°C.; 10 μl CD41 PE+5 μl CD62 FITC was added and mixed with Vortex;incubateed during 5 min. at room temperature; 1000 μl cold HBSS bufferwas added; measurement in flowcytometer was performed.

Platelet aggregation was measured according to Platelet AggregationProfiler PAP 8 (Mölab) using all reagents from Mölab.

Sample Preparation:

3 ml platelet concentrate was centrifuged at 3000 RPM to get PPP. 1800μl PPP and 600 μl PRP were mixed.

Test:

225 μl PRP was brought in test tube with magnetic stirrer. 225 μl PPP+25μl aquadest was brought in test tube without magnetic stirrer. PPP wasused for basis. PRP was put during 2 min at 37° C. on a roller.Measurement with PRP in aggregometer was started after 30 seconds with25 μl inductor. Measurement was performed during 6 min.

Visual determination of swirling was performed according to Bertolini,F. and Murphy, S. (1994) (A multicenter evaluation of reproducibility ofswirling in platelet concentrates, Transfusion 34, 796-801.)

Results of Tests with PRP Collected Platelets

PRP platelets were tested with the addition of Sul 136. Table 4 showsthe results of the number cells that survived 24, 48, 72, and 216 hoursafter storage at 4° C. The number of cells are given in % relative tothe number of cells at time 0. 66% of the platelets stored at roomtemperature survived after 216 hours. Only 42.3% of the cells stored at4° C. survived after 216 hours. The addition of Sul 136 resulted thatsubstantial more platelets survived after storage of 72 and 216 hours at4° C.

TABLE 4 Time in hours Sample 0 72 216 PRP Room Temp 100 80.7 66.1 PRP 4°C. 100 43.3 42.3 60 min 0.001 mM 100 83.8 86.1 60 min 0.01 mM 100 82.994.8 60 min 0.1 mM 100 76.3 77.6 60 min 1 mM 100 29.9 62.9 60 min 10 mM100 51.8 43.4 10 min 0.001 mM 100 58.9 55.1 10 min 0.01 mM 100 66.0 52.110 min 0.1 mM 100 55.9 50.8 10 min 1 mM 100 59.1 59.3 10 min 10 mM 10064.8 47.9

Table 5 shows the possibility of aggregation of the platelets collectedwith PRP and where Sul 136 was added, after stimulation with ADP. When10 mM Sul 136 was added, the platelets still showed activity afterstimulation with ADP. (+means that the cells show aggregation and canthus be activated upon stimulation, −means no aggregation uponstimulation)

TABLE 5 Sample 0 h 24 h 48 h 72 h 216 h PRP KT + + + − − PRP 4° C. + − −− − 60 min 0.001 mM + − − − − 60 min 0.01 mM + − − − − 60 min 0.1 mM + −− − − 60 min 1 mM + − − − − 60 min 10 mM + + + + − 10 min 0.001 mM + − −− − 10 min 0.01 mM + − − − − 10 min 0.1 mM + − − − − 10 min 1 mM + − − −− 10 min 10 mM + + + + +

Table 6 shows the possibility of aggregation of the PRP platelets withSul 136 after stimulation with collagen. When 10 mM Sul 136 was added,the platelets showed still activity after stimulation with collagen.(+means that the cells show aggregation, ++means strong aggregation,these platelets can thus be activated upon stimulation, −means noaggregation upon stimulation)

TABLE 6 Sample 0 h 24 h 48 h 72 h 216 h PRP KT + + + − − PRP 4° C. + − −− − 60 min 0.001 mM + − − − − 60 min 0.01 mM + − − − − 60 min 0.1 mM + −− − − 60 min 1 mM + − − − − 60 min 10 mM ++ ++ ++ ++ − 10 min 0.001 mM +− − − − 10 min 0.01 mM + − − − − 10 min 0.1 mM + − − − − 10 min 1 mM + −− − − 10 min 10 mM ++ ++ ++ ++ ++Results with Platelet Collected Via Apheresis

Table 7 shows the results of the test of the stored apherese bloodplatelets of donor 1(2611770) stored at room temperature on a flatbedshaker without Sul 136. The pH at day 12 was 6.3. The pH at day 19 was5.7

Table 8 shows the results of the tests of the stored apherese bloodplatelets of donor 1, stored at 4° C. with the addition of Sul 136. ThepH on day 12 was 6.2, the pH on day 19 was 5.9.

TABLE 7 Platelet aggregation Flow cytometry MPV Collagen ADP P-selectinAnnexin (% Date Platelets/μl fL (%) (%) MFI ADP TRAP positive) Day 11,316,000 8.2 35 10 1.99 6.54 12.8 3.13 Day 2 1,248,000 9.7 72 0 2.093.97 12.3 5.15 Day 3 1,400,000 9.0 27 0 2.17 4.00 11.7 5.72 Day 51,212,000 9.6 0 0 3.21 4.09 12.6 6.41 Day 8 1,216,000 9.6 0 0 3.47 4.5310.2 16.70 Day 10 1,324,000 8.8 0 0 3.00 3.82 7.85 14.22 Day 121,528,000 9.2 0 0 4.34 4.78 6.51 21.45 Day 19 1,328,000 9.0 0 0 3.893.74 3.67 84.53

TABLE 8 Platelet aggregation Flow cytometry MPV Collagen ADP P-selectinAnnexin (% Date Platelets/μl fL (%) (%) MFI ADP TRAP positive) Day 11,000,000 10.6 0 0 2.09 3.07 3.57 4.51 Day 2 1,192,000 9.5 0 0 3.04 3.283.78 14.48 Day 3 1,240,000 9.2 0 0 3.73 4.12 4.95 18.24 Day 5 1,232,0009.1 0 0 4.12 4.20 5.55 20.38 Day 8 1,352,000 9.3 0 0 4.82 4.83 5.5234.56 Day 10 1,228,000 9.6 0 0 4.48 4.46 5.52 43.52 Day 12 1,204,000 9.817 12 4.57 4.43 4.42 56.91 Day 19 1,068,000 8.8 32 31 2.92 2.74 2.7688.34

The flow cytometry tests were similar for the platelets stored at roomtemperature and the platelets stored at 4° C. with compound Sul 136.Moreover, the platelet aggregation upon stimulation with collagen andADP was restored at day 12 and day 19 for platelets stored with compoundSul 136. This result is comparable with the platelets stored at roomtemperature at day 1-3.

Table 9 and 10 show the results of the test performed with plateletsform Donor 2. The platelets from table 9 were stored at 4° C. withoutSul 136. The platelets from table 10 were stored at 4° C. with Sul 136.

TABLE 9 Platelet aggregation Flow cytometry MPV Collagen ADP P-selectinAnnexin (% Date Platelets/μl fL (%) (%) MFI ADP TRAP positive) Day 11,008,000 7.8 89 15 1.82 4.67 7.83 3.89 Day 2 792,000 6.5 83 36 2.505.62 7.18 10.83 Day 3 632,000 6.3 86 22 2.76 5.51 5.87 17.92 Day 5824,000 6.5 82 27 3.43 6.44 7.30 24.04 Day 8 816,000 6.5 77 19 3.35 5.287.12 22.52 Day 10 684,000 6.5 83 13 3.00 4.12 5.13 22.78 Day 12 732,0006.5 75 12 2.93 3.92 5.15 28.12 Day 19 868,000 6.5 24 9 2.81 2.90 3.5434.18

TABLE 10 Platelet aggregation Flow cytometry MPV Collagen ADP P-selectinAnnexin (% Date Platelets/μl fL (%) (%) MFI ADP TRAP positive) Day 11,004,000 10.1 0 0 1.67 2.12 1.88 5.98 Day 2 1,272,000 7.6 0 0 2.13 2.532.35 10.96 Day 3 1,220,000 7.8 4 0 2.59 3.21 2.76 19.56 Day 5 1,252,0008.0 0 0 2.95 3.19 3.16 32.11 Day 8 1,168,000 8.0 0 0 2.99 3.08 3.2245.71 Day 10 1,104,000 8.1 0 0 2.76 2.83 2.96 53.96 Day 12 1,032,000 8.65 0 2.63 2.62 2.73 62.97 Day 19 860,000 8.8 0 0 2.01 1.99 2.14 72.67

Table 11 and 12 show the results of the test performed with plateletsfrom Donor 3. The platelets from table 9 were stored at room temperaturewithout Sul 136. The platelets from table 10 were stored at 4° C. withSul 136.

TABLE 11 Platelet aggregation Flow cytometry MPV Collagen ADP P-selectinAnnexin (% Date Platelets/μl fL (%) (%) MFI ADP TRAP positive) Day 11,060,000 7.7 80 9 2.10 6.09 10.80 3.69 Day 2 1,004,000 9.6 81 0 2.183.81 9.20 3.87 Day 3 1,060,000 7.6 75 0 2.34 3.74 9.48 5.21 Day 5964,000 10.1 34 0 2.80 4.29 10.80 6.12 Day 8 956,000 9.6 72 0 3.55 4.7011.30 5.38 Day 10 976,000 9.3 23 0 3.30 4.36 8.44 8.09 Day 12 1,068,0009.2 41 0 3.67 4.85 7.39 14.08 Day 19 1,028,000 9.7 0 0 4.76 4.56 4.5990.52

TABLE 12 Platelet aggregation Flow cytometry MPV Collagen ADP P-selectinAnnexin (% Date Platelets/μl fL (%) (%) MFI ADP TRAP positive) Day 1980,000 10.3 0 0 1.90 2.71 2.08 5.14 Day 2 1,188,000 7.7 0 0 2.82 3.493.62 8.68 Day 3 1,072,000 7.7 0 0 3.84 4.96 4.42 14.34 Day 5 1,068,0008.0 0 6 4.50 4.84 5.04 27.77 Day 8 992,000 8.3 0 0 4.82 4.91 5.24 41.97Day 10 892,000 8.3 0 0 4.37 4.25 5.04 62.65 Day 12 912,000 9.0 8 0 3.893.84 4.20 70.33 Day 19 692,000 9.5 0 0 2.22 2.37 2.01 80.99

Swirling Test

Still after 7 weeks Swirling is observed in the platelets stored withthe compounds at 4 C

Swirling is not observed at platelets without added compounds stored at4 C, measured after 24 hours.

EXAMPLE 4: SYNTHESIS OF THE COMPOUNDS

The compounds according to the invention are synthesized according tostandard synthesis methods which are well known by a person skilled inthe art.

SUL-0083, SUL-0084 and SUL-0085 are commercially available.

Synthesis of SUL 089-112, 114-117, 120-126, 128-130, 132, 134-135, 138,and 140.

Amidation of trolox was achieved by reaction with the appropriate aminein the presence of standard coupling reagents for amide formation, e.g.,HATU and CDI. The corresponding amines were prepared by reduction of theamides formed with BH₃

Hydroxamic acid derivatives were prepared by reaction withhydroxylamine/CDI. The synthesis of carbohydrazide analogues of troloxwas achieved by reaction with (substituted) hydrazines.Enantiomeric/diastereomeric compounds were prepared starting fromenantiomerically pure (R)- or (S)-Trolox or by means of chiralchromatography.

Synthesis of SUL-118, SUL-119 en SUL-146

Oxidation of commercially available propofol with salcomine, acoordination complex of the salen ligand with cobalt, followed byreduction with NaBH₄ afforded 2,6-diisopropylbenzene-1,4-diol Subsequentmethylation with HCO/SnCl₂/HCl and reaction with methyl methacrylatefurnished SUL-146 (methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate). Hydrolysiswith LiOH yielded the carboxylic acid SUL-118(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid). Thealcohol SUL-119(2-(hydroxymethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol) wasobtained by reduction of SUL-146 with LiAlH₄.

Synthesis of SUL-131, SUL-133, SUL 137 en SUL-146

Starting from the carboxylic acid SUL-118(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid), thehydroxylamine was obtained by reaction with hydroxylamine using CDI ascoupling reagent. Compounds SUL 133((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone) and SUL 137((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(piperazin-1-yl)methanone) were prepared by reaction of SUL-118 with theappropriate piperazine derivative. Both coupling reagents HATU and CDIresulted in satisfactorily yields. SUL 139(2-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)aceticacid) was prepared by a reductive amination of SUL 137((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(piperazin-1-yl)methanone) with glyoxalic acid.

Synthesis of SUL-136, SUL-141 and SUL-142.

Hydrolysis of SUL-140 (ethyl2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetate)under N, atmosphere furnished SUL-136(2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)aceticacid) in high yield. The enantiomers SUL-141 and SUL-142 were preparedaccording to the above-described conditions.

Synthesis of SUL 143, 144 en 145

Amidation of trolox with (S)-methyl pyrrolidine-2-carboxylate (L-prolinemethyl ester) afforded, after column chromatography, twodiastereoisomers. Subsequent hydrolysis of the individualdiastereoisomers afforded SUL-144((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid, diastereomer 1) and SUL-145((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid, diastereomer 2). The racemic analogue SUL-143((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylicacid) was obtained by mixing the esters of the individualdiastereoisomers followed by hydrolysis of the ester moiety using LiOH.

Amidation of Trolox (General Example) SUL-108 ((4-butylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone)

HCl. Trolox (11 g, 0.044 mol, 1 eq.) was suspended in acetonitrile(100-150 ml). CDI (8.6 g, 0.053 mol, 1.2 eq.) was added in portions. Thereaction mixture was stirred for 0.5-1 hour at room temperature. Afteraddition of 1-butylpiperazine (6.9 g, 0.048 mol, 1.1 eq.) the reactionmixture was stirred at 25-30° C. over the weekend. The reaction mixturewas concentrated, H₂O (200 ml) was added and the aqueous layer wasextracted with EtOAc (4×). The combined organic layers were dried,filtered and concentrated. The crude product obtained was purified bycolumn chromatography (DCM/10% MeOH) affording the compound aimed for (9g product, 82% pure). Crystallization from EtOAc/heptanes affordedSUL-108 (6 g, 0.016 mol, 36% yield, 90% pure) as a white solid. Thematerial obtained was dissolved in DCM (50-100 ml). HCl (4 M in dioxane,8.8 ml, 0.0035 mol, 2.2 eq.) was added and the reaction mixture wasstirred at room temperature over the weekend. The mixture was filtered,rinsed with DCM, and dried to afford the HCl salt of SUL-108 (6.3 g,97-98% pure) as a white solid.

¹H-NMR (CDCl₃, in ppm): 0.93 (t, 3H), 1.38 (m, 2H), 1.58 (s, 3H), 1.67(m, 2H), 2.09 (s, 3H), 2.12 (s, 3H), 2.15 (s, 3H), 2.50-3.20 (m, 14H).M⁺=375.3

Reduction of Trolox Amides (General Example) SUL-128.(2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol)

HCl. BH₃.THF in THF (16 ml, 0.0156 mol, 2 eq.) was cooled to T=0° C. Asolution of SUL-112((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone;2.6 g, 0.0078 mol, 1 eq.) in THF (50 ml) was added drop-wise and thereaction mixture was refluxed for 1 hour and cooled to room temperatureovernight. The reaction mixture was cooled on an ice bath and HCl (6 M,25 ml) was added drop-wise. DCM (100 ml) was added and the layers wereseparated. The aqueous layer was extracted with DCM (3×). The combinedorg. layers were dried over K₂CO₃ until no gas formation was noticedanymore. The organic phase was filtered and concentrated. The crudeproduct was cooled on an ice bath, and NaOH (6M, 50 ml) was addeddrop-wise. After addition the reaction mixture was stirred for 1 hourand extracted with DCM (4×). The combined DCM layers were dried,filtered and concentrated to give 1.6 g crude product (20-40% pure). Thematerial was purified by column chromatography affording SUL-128 (300mg, 0.94 mmol, 12% yield, 90% pure). This was dissolved in DCM (10 ml)and cooled to T=0° C. (ice bath). HCl (4M in dioxane, 0.3 ml, 0.94 mmol,1.2 eq.) was added and the reaction mixture was stirred at roomtemperature overnight. The solid formed was filtered, washed with Et₂Oand dried to afford the HCl salt of SUL-128 (300 mg, 90% pure) as awhite solid (mixture of diastereomers).

¹H-NMR (CDCl₃, in ppm): 1.20-1.90 (m, 7H), 2.12 (s, 6H), 2.17 (s, 3H),2.20-2.90 (m, 9H), 3.4-3.65 (m, 2H). M⁺=320.1

Synthesis of SUL-118(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid)Synthesis of 2,6-Diisopropylcyclohexa-2,5-diene-1,4-dione

Propofol 100 g, 561 mmol) was dissolved in DMF (250 mL). The solutionwas cooled to 0° C. while stirring. Salcomine (16.6 g, 51 mmol; 9 mol %)was added and the resulting reaction mixture was stirred 112 h overnightwhile warming to room temperature. The reaction mixture was poured inwater (7 L). The resulting slurry was extracted with heptanes (5×1 L).The combined organic extracts were dried with Na₂SO₄. Concentration ofthe solution under vacuum afforded the crude2,6-diisopropylcyclohexa-2,5-diene-1,4-dione (62.5 g; 325 mmol; 58%yield) as an oil. The product was used in the next step without furtherpurification.

Synthesis of 2,6-Diisopropylbenzene-1,4-diol

Crude 2,6-diisopropylcyclohexa-2,5-diene-1,4-dione (62.5 g, 325 mmol)was dissolved in dichloromethane (300 mL) and methanol (100 mL). Thesolution was cooled to 0° C. with an ice bath. Sodium borohydride (4.5g, 182 mmol) was added in portions. After the addition was complete thereaction mixture was stirred at room temperature overnight. Acetone (150mL) was added to quench the excess of sodium borohydride. After 30minutes stirring 2N aq. HCl (200 mL) was added. After stirring for 45minutes the mixture was extracted with ethyl acetate (4×400 mL). Thecombined organic layers were dried with Na₂SO₄. Concentration of thesolution under vacuum afforded crude 2,6-diisopropylbenzene-1,4-diol (64g, 330 mmol) as a red oil in quantitative yield. The product was used inthe next step without further purification.

Synthesis of 3,5-Diisopropyl-2-methylbenzene-1,4-diol

A mixture of 2,6-diisopropylbenzene-1,4-diol (64 g, 0.33 mol),paraformaldehyde (9.8 g, 0.327 mol), SnCl₂ (217.9 g, 1.15 mol),concentrated aq. 37% HCl (0.6 L) and diisopropyl ether (2.5 L) washeated to reflux for 4 hours. After cooling to room temperatureovernight the biphasic mixture was separated. The aqueous layer wasextracted with TBME (2000 mL). The combined organic fractions werewashed with 1N aq. HCl (1000 mL), water (1000 mL) and brine (1000 mL).The organic fractions were dried with Na₂SO₄ and concentrated undervacuum to give a 50:35 mixture of3,5-diisopropyl-2-methylbenzene-1,4-diol and2,6-diisopropyl-3,5-dimethylbenzene-1,4-diol (61 g oil) according toGCMS analysis. Purification by chromatography on silica gel (1200 mL)eluting with ethyl acetate/heptanes=97.5:2.5 (4000 mL), 95:5 (4000 mL)gave 3,5-diisopropyl-2-methylbenzene-1,4-diol 6 (16.6 g, 79.8 mmol; 24%:83% pure) as an oil.

Synthesis of Methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate

3,5-diisopropyl-2-methylbenzene-1,4-diol (10.6 g, 50.9 mmol; 83% pure)was dissolved in methyl methacrylate (20 mL, 186 mmol). The solution wastransferred to a Teflon tube in a Berghof reactor. Aqueous formaldehyde(10 mL; 37% wt. solution, stabilized with 10-15% MeOH) was added and thereaction mixture was heated to 180° C. (internal temperature) in theclosed reactor for 5 hours while stirring. After cooling to ca. 40° C.The reaction mixture was poured in MeOH (200 mL) and the mixture wasconcentrated under vacuum. Purification by chromatography on silica gel(600 mL) eluting with ethyl acetate/heptanes=95:5 (5000 mL; TLC: Rf˜0.2;spot stained with iodine vapor) gave the desired pure product methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (10.0 g,31.3 mmol, 61%).

Synthesis of 6-Hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylicacid (SUL-118)

A mixture of purified methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (8.3 g, 25.9mmol) and lithium hydroxide monohydrate (4.3 g, 102.5 mmol; 4 eq.) inMeOH (100 mL), THF (100 mL) and water (25 mL) was heated for 30 minutesat ambient pressure while rotating with a rotary evaporator in a warmwater bath at 60° C. The organic solvents were evaporated under vacuum.Water (150 mL) was added to the residue, followed by acetic acid (10mL). A light orange mixture was obtained. Extraction with ethyl acetate(3×100 mL), drying of the combined organic fractions with Na₂SO₄ andconcentration under vacuum gave the crude product as an orange solid.The solids were stirred with tBME (150 mL). A beige solid precipitatedand an orange solution was obtained. Heptane (250 mL) was added and themixture was stirred for 15 minutes. The mixture was filtered over aglass filter. The residual solids were washed with heptanes (2×50 mL) onthe filter under suction. Drying of the solids under vacuum at 60° C.gave pure 6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylicacid (SUL-118) as an off-white solid (3.1 g, 10.13 mmol; 39%, 100%pure). ¹H-NMR (CDCl₃, in ppm): 1.38 (t, 12H), 1.52 (s, 3H), 1.87 (m,1H), 2.20 (s, 3H), 2.30 (m, 1H), 3.20 (m, 1H), 3.38 (m, 1H). M+=307.10

EXAMPLE 5. SYNTHESIS OF SUL 119(2-(HYDROXYMETHYL)-5,7-DIISOPROPYL-2,8-DIMETHYLCHROMAN-6-OL)

A solution of methyl6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (500 mg,1.56 mmol) in THF (12 mL) was added over 5 minutes with a syringe via arubber septum to LiAlH₄ (238 mg, 6.26 mmol; 4 eq.), pre-weighed in a dry3-mecked 100 mL round bottomed flask under inert nitrogen atmospherewhile stirring at room temperature. The exothermic addition of the esterwas accompanied with gas evolution. After the addition was complete theresulting grey suspension was heated to reflux. After 3 hours theheating was stopped and the reaction was quenched by dropwise additionof EtOAc (6 mL; exothermic). Water (5 mL) was added in small portions,followed by 2N HCl (2 mL) followed by EtOAc (25 mL). The mixture waspoured on Na₂SO₄ (ca. 50 g) and the slightly yellow organic layer wasseparated from the two-phase mixture. The aqueous phase was washed withEtOAc (50 mL) and the combined organic fractions were concentrated undervacuum to give the crude alcohol (530 mg) as a clear oil. Heptane (100mL) was added and after concentration under vacuum the2-(hydroxymethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol (248 mg, 0.85mmol, 54%, LCMS: 95.5% pure).

M+=293.2

EXAMPLE 6. SYNTHESIS OF SUL 139(2-(4-(6-HYDROXY-5,7-DIISOPROPYL-2,8-DIMETHYLCHROMAN-2-CARBONYL)PIPERAZIN-1-YL)ACETICACID)

SUL-137 (440 mg, 1.17 mmol, 1 eq.,) was dissolved in MeOH (50 ml) andglyoxalic acid (216 mg, 2.35 mmol, 2 eq.) was added. The resultingmixture was stirred for 1 hour at room temperature and, subsequently,NaBH₃CN (183 mg, 2.94 mmol, 2.5 eq.) was added. The reaction mixture wasstirred at room temperature overnight. Acetic acid (few ml) was addedand after stirring at room temperature for 0.5-1 hour, the reactionmixture was concentrated. The residue obtained was dissolved in EtOAc,washed with H₂O (2×), dried, filtered and concentrated to afford SUL-139(500 mg, 1.16 mmol, 98%, 91-92% pure) as a light yellow solid.

¹H-NMR (CD₃OD, in ppm): 1.33 (dd, 12H), 1.59 (s, 3H), 1.62 (m, 1H), 2.09(s, 3H), 2-5-3.0 (m, 7H), 3.1-3.6 (m, 4H), 3.81 (bs, 2H), 4.28 (bs, 2H).M⁺=433.2.

EXAMPLE 7. SYNTHESIS OF SUL 136(2-(4-(6-HYDROXY-2,5,7,8-TETRAMETHYLCHROMAN-2-CARBONYL)PIPERAZIN-1-YL)ACETICACID)

A 250 ml three-necked flask equipped with two septa (left and right) anda stopcock was charged with SUL-136 (15.5 g, 38.4 mmol) and THF/water(240 ml THF+80 ml water). The clear solution was stirred and degassedfor at least 30 minutes by argon-bubbling, using an inlet tube equippedwith a long syringe needle through the left septum; the right septum wasequipped with a short needle and functioned as outlet. The degassedsolution (which was maintained under argon) was cooled to 0° C. in anice-bath and solid anhydrous LiOH (2.3 g, 96 mmol, 2.5 eq.) was added inone portion. The resulting reaction mixture was stirred for 2 hours at0° C. after which is was neutralized by addition of a MeOH/water (3/1,v/v) slurry of Dowex-50WX8-200 ion-exchange resin; the final pH wasapprox 6. The Dowex resin was filtered off with suction and rinsed with3 portions of MeOH/water (3/1, v/v). The filtrate was reduced in vacuoand to the wet product was added approx. 100 ml water. The resultingwhite aqueous suspension was freeze-dried overnight to afford SUL-136(13.48 g, 93%. LCMS: 99.6%) as a white solid. 1H-NMR (CD3OD, in ppm)):1.60 (s, 3H), 1.65 (m, 1H), 2.05 (s, 3H), 2.10 (s, 6H), 2.55 (m, 2H),2.62 (m, 1H), 3.0, (bs, 4H), 3.40 (bs, 2H), 3.65 (bs, 2H), 4.25 (bs,2H). M+=377.1

EXAMPLE 8. SYNTHESIS OF SUL 144((2S)-1-(6-HYDROXY-2,5,7,8-TETRAMETHYLCHROMAN-2-CARBONYL)PYRROLIDINE-2-CARBOXYLICACID)

(2S)-methyl1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylate(diastereomer 1, 3.5 g, 9.7 mmol) was dissolved in THF/H₂O (60/20 mL).N₂ was bubbled through the solution for 1 h. The mixture was cooled inan ice-bath and LiOH.H₂O (1.01 g, 24.2 mmol, 2.5 eq.) was added. Thereaction mixture was stirred under N₂ at RT overnight. Dowex-50WX8-200(washed 4× with MeOH/H₂O 3:1) was added as a slurry in MeOH/H₂O (3:1)until the pH=6. The mixture was filtered, washed with MeOH/HO₂ (3:1) andconcentrated in vacuo. Demi H₂O (50 mL) was added to the concentrate andthe solution was freeze dried affording SUL-144 (3.4 g, 9.7 mmol, quant,99.7% pure) as a off-white foam.

1H-NMR (CDCl₃): 1.60 (s, 3H), 1.65-2.30 (m, 14H), 2.60 (m, 2H), 2.81 (m,1H), 3.49 (m, 1H), 4.01 (t, 1H), 4.50 (d, 1H)

M+=348.1

EXAMPLE 9: SUL 109 FOR COLD ISCHEMIC TOLERANCE OF PORCINE HEARTS

This example studies if SUL 109 as additive to standard hearttransplantation protocols will help to prolong the maximal cold ischemicprotection of porcine hearts. For long term arrest, for example in caseof transplantation, a commonly used solution is CUSTODIOL®. WithCUSTODIOL® solution it is possible to keep a heart in a cold ischemicstate for up to six hours before reperfusion with warm oxygenated bloodis necessary.

Material and Methods

Two hearts were harvested from slaughterhouse pigs and treated asfollows. Pigs were stunned by electrical shock to the head andexsanguinated by severing the superior caval vein. After exsanguinationsthe sternum was quickly opened and the heart and lungs were removed as awhole. The heart was immediately submersed in an ice cold bath and theaorta was cut proximal to the brachocephalic side branches. A 19 mmcannula was inserted in the aorta and tied off. The carefully de-airedcannulla was used to retrogradely administer cold cardioplegic solution.In the first heart, 2 liters of standard CUSTODIOL® with 5000 IU/1 ofheparin added was administered. The second heart was administered 2liters of CUSTODIOL® with 5000 IU/1 of heparin and 10 ml/l of SUL 109 in0.9% NaCl at 75 μM. Both hearts were stored in plastic bags filled withthe same solutions and transported to the lab on ice at approximately 4°C. Before preparation the hearts were stored at 4° C. for 24 hours. Thenext day both hearts were prepared to be mounted in the PhysioHeartplatform as described in DeHart et al 2011. After de-airing the hearts,retrograde reperfusion of the aorta was started with warm oxygenatedblood at 38° C. Both flow of blood and pressure at the aortic root wereregistered during the experiment.

Results and Findings

First the non SUL109 treated heart was reperfused with blood. This heartshowed high vascular resistance at reperfusion resulting in a totalcoronary blood flow of 0.5 liters per minute at a set aortic pressure of80 mmHg. Hardly any contractile activity of the heart muscle was visibleafter 5 minutes, only a slight motion of tissue resembling fibrillation.After some defibrillation by electric shock and assisting the heart witha pacemaker, a vague contraction of the lateral and anterior leftventricle was visible.

The second heart, which was treated with SUL109, was also reperfusedwith blood. Immediately after the start of reperfusion, the firstremarkable difference with the non treated heart occurred. The muscletissue of the treated heart initially was hard and stiff as the nontreated heart but at reperfusion and warming up the heart graduallybecame less hard and stiff and almost feeling like a normal heart atreperfusion within 6 hours. This resulted in a lower vascularresistance, as observed by a coronary flow of 1 lpm at a perfusionpressure of 80 mmHg. The treated heart immediately displayed morecontractile activity compared to the non treated one and after somedefibrillation shocks a weak contractive unpaced pattern was observed.

Discussion

In the normal harvesting procedure of the PhysioHeart experiments, thehearts are arrested on a cold cardioplegic solution and transported tothe LifeTec Group laboratories for further preparation and surgicalinterventions. Preparations can take up to 4 hours after which the heartis reactivated by reperfusion with warm oxygenated blood. In order toincrease the preparational time, or to allow longer travel time beforereperfusion, it would be very useful to be able to protect myocardialtissues during the cold ischemic time. This example shows a cleardifference observed between the SUL109 treated heart and the non-treatedheart, as the former showed an improved regular contractive activity ofthe myocardial tissue. Accordingly, using SUL 109 as an additive tostandard heart transplantation protocols prolongs the cold ischemicprotection.

1-9. (canceled)
 10. Method for protection of cells comprising adding acompound to a cell, wherein the compound has the structural formula of(I)

wherein, R1, and R2 are independently selected from the group consistingof C₁-C₆ alkyl and is preferably methyl, ethyl, propyl or isopropyl; R3is selected from the group consisting of —CH₂OH, CH₂NHR₉, C(═O)YR₁₀,

where * indicates the point of attachment of R₃ to the remainder of themolecule; R₄, R₅, R6, R₇, R₈ are independently selected from the groupconsisting of H, —OH, alkyl, substituted alkyl, preferably hydroxyalkyl,aryl, substituted aryl, halogen, oxygen, heteroaryl, substitutedheteroaryl; X is selected from the group consisting of H, O, S; Y isselected from the group consisting of O, NH, S; R₉ is selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, preferablyhydroxyalkyl or substituted hydroxyalkyl, alkylbenzylfluoride, alkenyl,aryl, substituted aryl, preferably haloaryl, heteroaryl; R₁₀ is selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl,preferably hydroxyalkyl or cyanoalkyl, haloalkyl, alkylamide,substituted alkylamide, aryl, substituted aryl, preferably nitrobenzyl,halobenzyl, alkylbenzoyl, OH, alkenyl, alkadienyl, alkylhalide,arylhalide, —CH₂(C═O)O-alkyl, heteroaryl, substituted heteroaryl,—NH—CH₂CH₂CN; R₁₁ or R₁₂, alkyl, substituted alkyl, preferably analkylamine, or form together with the atom N to which they are attacheda saturated or unsaturated 3-8 membered ring, optionally incorporatingone or more additional, such as one, two, or three N, O, or S atoms,optionally substituted with an alkyl, alkylalcohol; R₁₃ and R₁₄ togetherwith the atom N to which they are attached form a saturated orunsaturated 3-8 membered ring, optionally incorporating one or moreadditional, such as one, two, or three N, O, or S atoms, optionallysubstituted, preferably with an alkyl, alkylalcohol.
 11. Methodaccording to claim 10, wherein the protection of cells occurs duringstorage.
 12. Method according to claim 11, wherein the storage occurs ata temperature below 37° C., in particular at room temperature, at about4° C., at about −20° C. or at about −80° C.
 13. Method according toclaim 10, where the addition of the compound occurs prior to cooling thecells.
 14. Method according to claim 10, wherein the cells are mammaliancultured cells, mammalian primary cells or blood platelets.
 15. Methodaccording to claim 10, wherein the cells are blood platelets and thecompounds are Sul 100, 117, 118, 125, 126, 132, 136, 138, 139, 141, 142,143, 144,
 145. 16. (canceled)